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Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analyses
Sevim Kahraman1,2,3, Kimitaka Shibue1,2,3, Dario F De Jesus1,2,3
1Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, United States.
Elife
|September 21, 2023
Summary
Researchers developed a simple method using DA-ZP1 to purify human pancreatic alpha-cells (~95% purity). These purified cells form viable pseudoislets for studying glucagon secretion and gene expression in glucose homeostasis.
Area of Science:
- Endocrinology
- Cell Biology
- Biochemistry
Background:
- Pancreatic alpha-cells produce glucagon, essential for glucose homeostasis.
- Isolating pure human alpha-cells for research is difficult due to purification challenges.
Purpose of the Study:
- To develop a cost-effective and efficient method for purifying live human pancreatic alpha-cells.
- To establish a method for culturing purified alpha-cells for functional and transcriptional studies.
Main Methods:
- Utilized the reaction-based probe diacetylated Zinpyr1 (DA-ZP1) for cell enrichment.
- Employed cell sorting and immunostaining to confirm alpha-cell purity and identity.
- Cultured sorted alpha-cells to form pseudoislets for up to 10 days.
Main Results:
- Achieved ~95% purity of live human alpha-cells using the DA-ZP1 method.
- Established viable alpha-pseudoislets that maintained glucagon secretion in response to glucose.
- RNA-sequencing showed sustained expression of key alpha-cell genes in culture, with time-dependent alterations in specific genes like DLK1.
Conclusions:
- A novel, simple, and high-purity method for isolating human primary alpha-cells was established.
- The method enables downstream functional and transcriptional analyses of human alpha-cells.
- Cultured alpha-pseudoislets provide a valuable model for studying alpha-cell biology.
